Construction of a Stable Replicating Shuttle Vector for <i>Caldicellulosiruptor</i> Species: Use for Extending Genetic Methodologies to Other Members of This Genus

<div><p>The recalcitrance of plant biomass is the most important barrier to its economic conversion by microbes to products of interest. Thermophiles have special advantages for biomass conversion and members of the genus <i>Caldicellulosiruptor</i> are the most thermophilic cellulolytic microbes known. In this study, we report the construction of a replicating shuttle vector for <i>Caldicellulosiruptor species</i> based on pBAS2, the smaller of two native <i>C. bescii</i> plasmids. The entire plasmid was cloned into an <i>E. coli</i> cloning vector containing a pSC101 origin of replication and an apramycin resistance cassette for selection in <i>E. coli</i>. The wild-type <i>C. bescii pyrF</i> locus was cloned under the transcriptional control of the regulatory region of the ribosomal protein S30EA (Cbes2105), and the resulting vector was transformed into a new spontaneous deletion mutant in the <i>pyrFA</i> locus of <i>C. bescii</i> that allowed complementation with the <i>pyrF</i> gene alone. Plasmid DNA was methylated <i>in vitro</i> with a recently described cognate methyltransferase, M.CbeI, and transformants were selected for uracil prototrophy. The plasmid was stably maintained in low copy with selection but rapidly lost without selection. There was no evidence of DNA rearrangement during transformation and replication in <i>C. bescii</i>. A similar approach was used to screen for transformability of other members of this genus using M.CbeI to overcome restriction as a barrier and was successful for transformation of <i>C. hydrothermalis,</i> an attractive species for many applications. Plasmids containing a carbohydrate binding domain (CBM) and linker region from the <i>C. bescii celA</i> gene were maintained with selection and were structurally stable through transformation and replication in <i>C. bescii</i> and <i>E. coli</i>.</p></div

Strains and plasmids used in this work.

1<p>German Collection of Microorganisms and Cell Cultures.</p

Chromosomal map and PCR analysis of the Uridine Monophosphate (UMP) biosynthetic gene cluster in <i>C.bescii</i> DSM 6725 and the spontaneous deletion in <i>pyrFA</i> (JWCB005) locus.

<p>(A) A diagram of the <i>pyr</i> operon region with the 878 bp deletion in the <i>pyrFA</i> ORFs. The line below the diagram indicates the length of the deletion. Bent arrows depict primers used for verification of the structure of the chromosome in the JWCB005 (Δ<i>pyrFA</i>) strain. <i>pyrF</i> and <i>pyrE</i> loci indicated as black color filled arrow and black dashed filled arrow, respectively. (B) Gel depicting PCR products of the <i>pyrFA</i> region in wild type (3.44 kb) compared to the Δ<i>pyrFA</i> (2.52 kb) strain amplified by primers (JH020 and FJ298). (C) Gel depicting the 2.66 kb PCR products of <i>pyrE</i> region in wild type and the Δ<i>pyrFA</i> strain by primers (DC326 and DC331). M: 1 kb DNA ladder (NEB).</p

Plasmid map of shuttle vector (pDCW89) and verification of its presence in <i>C.bescii</i> transformants.

<p>(A) A linear DNA fragment containing the <i>pyrF</i> expression cassette as well as the entire sequence of pBAS2, generated by PCR amplification using primers DC283 and DC284, was ligated to a DNA fragment containing <i>E. coli</i> replication and selection functions to generate the final shuttle vector. The cross-hatched box corresponds to the pBAS2 plasmid sequences. ORFs from <i>C. bescii</i> are indicated as empty arrows and those from <i>E. coli</i> as black arrows. The apramycin resistant gene cassette (Apr^R); PSC101 low copy replication origin in <i>E. coli</i>; <i>repA</i>, a plasmid-encoded gene required for PSC101 replication; <i>par</i>, partition locus are indicated. The proposed replication origin (115 bp) of pBAS2 is indicated. The primers and restriction sites (AatII and EcoRI) used for the verification are indicated. A detailed description of the construction of pDCW89 is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062881#pone.0062881.s001" target="_blank">Fig. S1</a> and the Materials and Methods. (B) Gel showing the 1.6 kb PCR products containing the pSC101 <i>ori</i> sequences only presence in pDCW89 using primers DC230 and JF199, total DNA from JWCB005 (Lane 1), a <i>C. bescii</i> transformant with pDCW89 (Lane 2), and pDCW89 isolated from <i>E. coli</i> (Lane 3) as template. (C) Restriction analysis of plasmid DNA before and after transformation of <i>C. bescii</i> and back-transformation to <i>E. coli</i>. Lanes 1 and 4, pDCW89 plasmid DNA isolated from <i>E. coli</i> DH5α, and digested with AatII (Lane 1, 4.4 kb and 3.3 kb cleavage products), and EcoRI (Lane 4, 1.9 kb and 5.8 kb cleavage products); lane 2, 3, 5, 6, plasmid DNA isolated from two biologically independent <i>E. coli</i> DH5α back-transformed from <i>C. bescii</i> transformants, and digested with AatII (Lane 2 & 3), and EcoRI (Lane 5 & 6). M: 1 kb DNA ladder (NEB).</p

Comparison of DNA modification status between shuttle vector DNA isolated from <i>E.coli</i> (Lane 1) and <i>C. hydrothermalis</i> transformants (Lane 2) by Restriction analysis.

<p>(A) Undigested, (B) Digested with HindIII (4.3 and 3.4 kb cleavage products); (C) Digested with EcoRI (4.6 and 1.9 kb cleavage products); (D) Digested with CbeI (11 cleavage products are expected). M: 1 kb DNA ladder (NEB).</p

MOESM1 of Cellulosic ethanol production via consolidated bioprocessing at 75 °C by engineered Caldicellulosiruptor bescii

Additional file 1: Figure S1. The diagram for Teth39_0206 (adhE) expression cassette integration vector in C. bescii. Figure S2. The diagram for Teth39_0218 (adhB) expression cassette integration vector in C. bescii. Table S1. List of primers used in this study

Additional file 1: of Expression of the Acidothermus cellulolyticus E1 endoglucanase in Caldicellulosiruptor bescii enhances its ability to deconstruct crystalline cellulose

Figure S1. Plasmid map of chromosomal knock-in vector in C. bescii for extracellular expression of E1 (Acel0614). Figure S2. Relative quantification of enzymatic activity of the extracellular fraction of C. bescii expressing E1 (Acel0614) on Avicel and carboxymethylcellulose. Table S1. The list of Glycoside Hydrolase Family 5 (GH5) catalytic domains in Caldicellulosiruptor bescii and their sequence homology with the GH5 domain in E1 from A. cellulolyticus. Table S2. Primers used in this study

Sustainable Production of Shinorine from Lignocellulosic Biomass by Metabolically Engineered Saccharomyces cerevisiae

Mycosporine-like amino acids (MAAs) have been used in cosmetics and pharmaceuticals. The purpose of this work was to develop yeast strains for sustainable and economical production of MAAs, especially shinorine. First, genes involved in MAA biosynthetic pathway from Actinosynnema mirum were introduced into Saccharomyces cerevisiae for heterologous shinorine production. Second, combinatorial expression of wild and mutant xylose reductase was adopted in the engineered S. cerevisiae to facilitate xylose utilization in the pentose phosphate pathway. Finally, the accumulation of sedoheptulose 7-phosphate (S7P) was attempted by deleting transaldolase-encoding TAL1 in the pentose phosphate pathway to increase carbon flux toward shinorine production. In fed-batch fermentation, the engineered strain (DXdT-M) produced 751 mg/L shinorine in 71 h. Ultimately, 54 mg/L MAAs was produced by DXdT-M from rice straw hydrolysate. The results suggest that shinorine production by S. cerevisiae might be a promising process for sustainable production and industrial applications